Chemosphere 61 (2005) 218–228 www.elsevier.

com/locate/chemosphere

Role of ethylene diurea (EDU) in assessing impact of ozone on Vigna radiata L. plants in a suburban area of Allahabad (India)
S.B. Agrawal
a b

a,*

, Anoop Singh b, Dheeraj Rathore

b

Lab of Air Pollution and Global Climatic Change, Department of Botany, Banaras Hindu University, Varanasi 221 005, India Lab of Air Pollution and Global Climatic Change, Department of Botany, Allahabad Agricultural Institute—Deemed University, Allahabad 211 007, India Received 24 July 2003; received in revised form 12 November 2004; accepted 27 January 2005 Available online 13 March 2005

Abstract A field study was conducted to evaluate the suitability of ethylene diurea (N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N 0 phenylurea; EDU) in assessing the impact of O3 on mung bean plants (Vigna radiata L. var. Malviya Jyoti) grown in suburban area of Allahabad city situated in a dry tropical region of India. EDU is a synthetic chemical having anti-ozonant property. Mean monthly O3 concentration varied between 64 and 69 lg mÀ3 during the experimental period. In comparison to EDU-treated plants, non-EDU-treated plants showed significant reductions in plant growth and yield under ambient conditions. Significant favourable effects of EDU-application were observed with respect to photosynthetic pigments, soluble protein, ascorbic acid and phenol contents. EDU-treated plants maintained higher levels of pigments, protein and ascorbic acid in foliage as compared to non-EDU-treated ones. The study clearly demonstrated that EDU alleviates the unfavourable effects of O3 on mung bean plants, and therefore can be used as a tool to assess the growth and yield losses in areas having higher O3 concentrations. Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: EDU; Urban air pollution; Ozone; Growth; Yield; Vigna radiata

1. Introduction Rapid increase in urbanization and industrialization in many developing countries during last three decades has led to significant increases in atmospheric concentrations of primary and secondary air pollutants (Slanina et al., 1995; Mage et al., 1996; UNEP, 1999). In India, urban air pollution problems have increased tremendously due to increase in urban population, number of
Corresponding author. Tel.: +91 542 2368156; fax: +91 542 2368174. E-mail addresses: sbagarwaldr@sancharnet.in, sbagrawal@ sify.com (S.B. Agrawal).
*

motor vehicles, use of fuels with poor environmental performance, badly maintained roads, and ineffective environmental regulations. As a result agricultural lands adjacent to urban areas are increasingly exposed to air pollutants of urban origin. These phytotoxic gases may have significant impacts on the livelihoods and well beings of poor farmers and consumers through their effects on urban and periurban crop production (Te Lintelo et al., 2002). Elevated concentrations of tropospheric O3 at peri-urban and urban sites of major cities of developing countries, including Pakistan (Ghauri et al., 1991; Wahid et al., 1995, 2001), Indonesia (Komala and Ogawa, 1991; Pardede, 1991) and India (Agrawal et al., 2003) have been reported. For Asia, including

0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.01.087

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India and Eastern China, maximum mean of three monthly O3 concentrations have been projected to vary between 60 and 70 ppb for the year 2030 based on a global ozone model assuming a ‘‘business as usual’’ scenario (Emberson et al., 2003). Ambient levels of O3 have been reported to cause foliar injury and reduce productivity of a number of crop plants. Effects of elevated O3 concentrations include a decrease in plant growth, an alteration in plant metabolism and ultimately reduction in crop yield (Fumagalli et al., 2003). Various experimental studies have clearly demonstrated the deleterious effects of elevated O3 in potato (Finnan et al., 2002), wheat (Wahid et al., 1995; Gelang et al., 2001), rice (Maggs and Ashmore, 1998), tobacco (Nakajima et al., 2002) and soybean (Wahid et al., 2001), and snap bean (Tonneijck and Van Dijk, 2002a,b). EDU (N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N 0 -phenyl urea (abbreviated EDU for ethylenediurea) is known to suppress acute and chronic injury due to O3 in a wide range of plants without appreciable effects of its own (Carnahan et al., 1978; Bortier et al., 2001; Agrawal, 2002; Manning et al., 2003). EDU-treatment led to a significant increase in biomass and yield of bean plants exposed to O3 in open top chambers (Brunschon-Harti et al., 1995a). Studies have also been conducted on the mode of action of EDU (Lee et al., 1997; Gillespie et al., 1998; Godzik and Manning, 1998; Agrawal and Agrawal, 1999). The use of EDU, however, is still a controversial matter because the exact mechanism of action is unknown, and the chemical is known to be toxic at higher concentrations (Eckardt and Pell, 1996). Nevertheless, it has been suggested that EDU may be an useful ‘‘experimental tool’’ to determine the location and magnitude of crop losses due to O3, but not as a protecting agent against O3 induced injury and crop losses. In India, meteorological conditions such as high temperature and high light intensity with long light duration are favourable to O3 formation due to long range transport of precursor emissions. Use of EDU as a research tool may be economical as very few laboratories in India are equipped with advanced O3 gas monitors, and even it may be used without electricity in remote areas. In the present study, therefore, an effort has been made to ascertain the suitability of EDU as soil drench to assess the impacts of ambient O3 on mung bean plants (Vigna radiata L. var. Malviya Jyoti) grown under field conditions in a suburban area of Allahabad city (India) experiencing higher ambient O3 levels. Selected biochemical parameters were also studied to explain the EDU-induced O3 protection.

2. Materials and methods The study was performed in suburban area of Allahabad city located in the dry tropical Gangetic plains of

India at 24°47 0 N latitude, 82°21 0 E longitude and 96 m above mean sea level. Annual average temperature was 24 °C, relative humidity was 65% and annual precipitation was 959 mm. There were two field sites (i) AAI: agricultural field of Allahabad Agricultural Institute, and site (ii) Ar: agricultural field in Arail area. Site AAI is situated downwind of a national highway connecting states of Uttar Pradesh and Madhya Pradesh. The traffic on highway is dominated by heavy commercial vehicles. Site Ar was selected 16 km upwind from AAI near river Jamuna having mean 8 hourly O3 concentration between 11.6 and 15.2 g mÀ3 during experimental period. The field plots were prepared for seed sowing by ploughing to a depth of 0.25 m after mixing farmyard manure. Seeds of mung bean were sown in 6 plots (1.0 · 1.0 m size) at a distance of 5 cm in rows. After germination, plants were thinned to one plant every 20 cm in each row. Three hand weedings were done during 60 days after sowing (DAS) depending upon the weed intensity. After one week of seedling emergence three plots were treated with EDU (500 ppm) solution at weekly interval up to 80 days. EDU solution was freshly prepared in deionized water and 500 ml plantÀ1 was applied as a soil drench at 7:30 AM. Control plants received only deionized water (500 ml plantÀ1). Plants were subjected to identical water regime both for EDU-treated and non-EDU-treated ones. Random samples of plants were taken in triplicate at 30, 45, 60 and 75 days after sowing (DAS) for various analyses. For the estimation of chlorophyll and carotenoid contents, 0.1 g leaf sample was placed in 10 ml cold 80% acetone in a stoppered tube for overnight at 4 °C in a refrigerator. They were then homogenized and centrifuged at 6000 · g for 15 min. Optical densities of leaf extract were taken at 480 and 510 nm wavelengths for carotenoids and 645 and 663 nm wavelengths for chlorophyll on a UV–Vis-Spectrophotometer (Systronics, model 117, India). The amounts of chlorophyll a and b were calculated by using the formulae developed by Maclachalan and Zalik (1963), and of carotenoid by Duxbury and Yentsch (1956). For protein extraction fresh leaves were homogenized in tris buffer (0.1 M) followed by mixing of TCA (10%) and then dissolved into 0.1 N NaOH. For estimation of protein, method of Lowry et al. (1951) was followed. For ascorbic acid, leaf sample was homogenized in oxalic acid and Na EDTA extraction solution. 2,6-dichlorophenolindophenol dye was used to develop colour and the absorbance was taken at 520 nm. After bleaching the colour by 1% ascorbic acid, the difference between absorbance was used to determine the ascorbic acid content as described by Keller and Schwager (1977). Phenol content was determined in acetone extract by using Folin-ciocalteu reagent and Na2CO3 (Bray and Thorpe, 1954).

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S.B. Agrawal et al. / Chemosphere 61 (2005) 218–228 Table 1 Ozone concentration in ambient air during experimental period (Mean ± 1SE) Site Concentration (lg mÀ3) July, 2002 Ar AAI 15.25 ± 0.46 64.00 ± 1.67 August, 2002 13.38 ± 0.86 69.00 ± 0.97 September, 2002 11.60 ± 0.67 66.50 ± 1.82

Morphological characteristics were analyzed with respect to root and shoot lengths, number of leaves and leaf area and number of nodules and pods. For total biomass estimation, plants were oven dried at 80 °C for 24 h and then weighed. Various growth indices were calculated using standard formulae of Hunt (1982). After final harvesting at 80 DAS, yield parameters such as seed weight podÀ1, yield plantÀ1, test weight (1000 seed weight) and harvest index (HI) were recorded. Harvest index was calculated as follows: HIð%Þ ¼ ðEconomic yield=Biological yieldÞ Â 100 Portable gas sampler was used to monitor O3 concentration for 8 h daily (8 AM to 4 PM) at weekly interval at both the experimental sites. Ozone was scrubbed in buffered KI (0.1 N) and the absorbing solution was immediately analyzed by the method of Byers and Saltzman (1958). Data were analyzed through one-way and two-way ANOVA test through SPSS software (SPSS Inc., version 10.0) for assessing the significance of quantitative changes in different parameters due to EDU treatment at different sampling intervals.

3. Results Ozone concentration ranged from 64 to 69 lg mÀ3 at site AAI, and from 11.6 to 15.2 lg mÀ3 at Ar (Table 1). Various growth, yield and biochemical parameters did not vary significantly between EDU-treated and nonEDU-treated plants at site Ar, which clearly depict that EDU itself does not have any effect on plants at given dose. Therefore, results of Ar site experiencing very low O3 levels have not been included here. Only results

obtained at site AAI experiencing high O3 levels are given in this communication. Photosynthetic pigments increased due to EDU application at all sampling intervals (Fig. 1). As compared to non-EDU-treated plants, chlorophyll a, b and total increased by 14.6%, 5.2% and 12.8%, respectively at 60 DAS in EDU-treated ones (Fig. 1A–C). Chlorophyll a, b and total and carotenoid contents varied significantly due to EDU-treatment and plant age (Table 2). A reduction of 9.7% in carotenoid content was observed in non-EDU-treated plants as compared to EDU-treated ones at 45 DAS (Fig. 1D). Ascorbic acid and protein contents increased significantly in EDU-treated plants than non-treated ones, while a reverse trend was observed for phenol content (Fig. 2). Statistical analyses showed that variations in foliar ascorbic acid, protein and phenol contents were significant due to age, EDU-treatment and their interactions (Table 2). Maximum increase in ascorbic acid (13.8%) and protein (9.8%) content was recorded in EDU-treated plants at 45 DAS (Fig. 2A and B). Phenol content reduced by 19.3% in EDU-treated plants at 45 DAS as compared to non-EDU-treated ones (Fig. 2C). Growth parameters also showed positive influence of EDU application as evidenced by increments in plant

1.75 1.50 1.25

Non-EDU EDU-treated

(A)

(C)

2.25 2.00 1.75

mg g-1 dry leaf

0.75 0.6 0.5 0.4 0.3 0.2 30 45 60

1.25

(B)

(D)

1.00 0.75 0.50 0.25 0.00

30

45

60

DAS

DAS

Fig. 1. Effect of EDU treatment on chlorophyll a (A), chlorophyll b (B), total chlorophyll (C) and carotenoid (D) contents of mung bean plants.

mg g-1 dry leaf

1.00

1.50

S.B. Agrawal et al. / Chemosphere 61 (2005) 218–228 Table 2 Significance level for various growth parameters and growth indices of mung bean plants as obtained by ANOVA test Parameters Two-way ANOVA Plant age Chlorophyll a Chlorophyll b Total Chlorophyll Carotenoid content Ascorbic acid Protein content Phenol content Plant height Root length Fresh shoot weight Fresh root weight Dry shoot weight Dry root weight Total biomass Leaf area Leaf number Nodule number Pod number Dry pod weight Root–shoot ratio (RSR) Leaf weight ratio (LWR) Specific leaf area (SLA) Net primary productivity (NPP) Absolute growth rate (AGR) Crop growth rate (CGR) Relative growth rate (RGR) Net assimilation rate (NAR) Leaf area index (LAI) Leaf area duration (LAD) *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ** *** Treatment *** *** *** ** *** *** *** *** *** *** *** * *** ** * *** *** *** *** *** NS NS ** * NS NS NS NS *** Plant age · treatment NS *** NS NS *** ** *** NS NS * * NS * NS NS NS *** *** *** ** NS NS NS NS NS NS NS NS NS

221

* = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = not significant.

height, biomass and leaf area of plants over non-EDUtreated ones (Fig. 3). An increase of 12.3% in plant height was recorded in EDU-treated plants at 80 DAS (Fig. 3A). Root length also increased in EDU-treated plants (Fig. 3B). Plant height, root length, fresh and dry shoot and root weights and total leaf area varied significantly (p < 0.001) due to age and EDU-treatment (Table 2). Fresh and dry biomass of root and shoot were higher in EDU-treated plants than non-EDU-treated ones (Fig. 3C–F). Total biomass increased by 24% due to EDU treatment at 60 DAS (Fig. 3G). Leaf area increased due to EDU-treatment, and a maximum increase of 30% was recorded at 80 DAS (Fig. 3H). Number of leaves, nodules and pods increased in EDU-treated plants than non-EDU-treated ones (Table 3). Number of leaves increased by 18.2%, 23.8%, 21.9% and 16.8% in EDU-treated plants at 30, 45, 60 and 80 DAS, respectively (Table 3). Two-way analysis of variance test showed highly significant (p < 0.001) variations in number of nodules and pods and dry pod weight due to plant age, EDU-treatment and their interactions

(Table 2). Number of nodules and pods and dry pod weight increased by 71.4%, 52.4% and 26.4%, respectively in EDU-treated plants at 80 DAS (Table 3). Higher RSR was observed in EDU-treated plants at all ages with maximum increase of 25% at 30 DAS (Fig. 4A). Significant effect of plant age (p < 0.001), EDUtreatment (p < 0.001) and their interaction (p < 0.01) was observed on RSR (Table 2). LWR was higher during first two sampling in EDU-treated plants and thereafter declined over non-EDU-treated ones. SLA showed a trend reverse of LWR (Fig. 4B–D). LWR and SLA varied significantly due to plant age (Table 2). NPP increased by 27.8% in EDU-treated plants than nonEDU-treated ones at 60 DAS (Fig. 4D). Variations in NPP were significant due to age (p < 0.001) and EDUtreatment (p < 0.01) (Table 2). AGR increased due to EDU-treatment with maximum increase of 66.7% during 60–80 DAS (Fig. 5A). AGR varied significantly due to age (p < 0.001) and EDU-treatment (p < 0.01) (Table 2). CGR and RGR of EDU-treated plants were higher during first two

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0.80 0.70 Non-treated EDU-treated

Ascorbic acid (mg g-1 fresh leaf)

4. Discussion In Indian cities, air pollutants concentrations often exceed the toxic limits set by Central Pollution Control Board (1997). These standards are available for SO2, NO2 and SPM, primarily based on human health impact, and not ideal for assessing pollutant impacts on vegetation. There is no ambient air quality standard for O3. High levels of automobile emission in urban areas of Allahabad cause reductions in plant growth and yield in suburban and urban areas (Singh et al., 2003). Annual average O3 concentration of 54 lg mÀ3 at an urban area of Pune and ten-days average of 30 lg mÀ3 at Nilgiri Biosphere forests located in South India were reported (Khemani et al., 1995). Pandey and Agrawal (1994) reported O3 concentration variations between 43 and 68 lg mÀ3 in Varanasi, an adjoining district of Allahabad. Agrawal et al. (2003) reported 6 h mean O3 concentration of 58.5 ppb in an area situated in the downwind of Varanasi during summer months of 1999–2000. In the present study, ambient O3 concentration at site AAI is comparable to the concentrations reported by Pandey and Agrawal (1994), Maggs and Ashmore (1998) and Nakajima et al. (2002). The site Ar showed very low O3 concentration as it was situated away from national highway in upwind direction and has extensive plantations. EDU has been successfully used as an alternative of open top chamber to assess crop loss due to O3 under ambient field conditions (Manning and Krupa, 1992; Hassan et al., 1995; Bell and Marshall, 2000; Wahid et al., 2001). Visible injury symptoms were not recorded in any of the EDU-treated and non-treated plants at AAI site. EDU concentration used during the present study was found effective in preventing O3 injury to a wide range of herbaceous plants (KostcaRick and Manning, 1993; Manning, 2000; Tonneijck and Van Dijk, 2002b; Manning et al., 2003). Pretreatment of EDU may have counteracted the formation of oxyradicals due to elevated O3 leading to maintenance of higher levels of photosynthetic pigments for longer period (Lee et al., 1997). Eckardt and Pell (1996) also found higher chlorophyll in EDU-treated potato plants than non-EDU-treated ones. Increase in carotenoids under EDU application had beneficial effects due to their protection to chlorophyll from photooxidative damage. Soluble protein and ascorbic acid contents increased, while phenol content decreased in EDU-treated plants. A similar result was reported by Brunschon-Harti et al. (1995b) for EDU-treated Phaseolus vulgaris L. plants. Ozone an oxidative stresser induces degradation of many biologically important molecules, such as amino acids, proteins and carbohydrates due to release of malondialdehyde (Janero, 1990; Alaiz et al., 1999). Lee and Chen (1982) found that EDU preserved protein

(A)
0.60

0.35

0.30 44

Protein (mg g-1 fresh leaf)

(B)
42

40 30 25 20 15 10 45

Phenol (mg g-1 fresh leaf)

(C)

40 10

5

0 30 45 DAS 60

Fig. 2. Effect of EDU treatment on ascorbic acid, protein and phenol contents of mung bean plants.

samplings and thereafter declined than the non-EDUtreated ones. NAR and LAR also showed a similar pattern (Fig. 5B–E). CGR, RGR, NAR and LAR varied significantly due to plant age (Table 2). Higher LAD was recorded in EDU-treated plants at all age intervals, with maximum during 60-80 DAS (Fig. 5F). LAD varied significantly (p < 0.001) due to age and EDU treatment (Table 2). Yield parameters showed positive influence of EDUtreatment (Fig. 6). Seed weight podÀ1 and yield plantÀ1 increased due to EDU application (Fig. 6A and B). Yield of EDU-treated plants increased by 32.2% as compared to non-EDU-treated ones. Harvest index was 16.9% and 13.4%, respectively for EDU-treated and nonEDU-treated plants (Fig. 6C). Test weight was 25.3% higher in EDU-treated plants than non-treated ones (Fig. 6D). Seed yield and test weight of plants varied significantly due to EDU-treatment (p < 0.05) (Table 4).

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Non-treated EDU-treated 20

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Plant height (cm)

80 60 40 20 0

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20 10 0 15
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Dry shoot weight (g)

10

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0.5

0 15 10 5 0
30 45 60 80 30 45 60 80

0.0 1500

Total biomass (g)

(G)

(H)
1000

500

0

DAS

DAS

Fig. 3. Effect of EDU treatment on selected growth parameters of mung bean plants. Table 3 Effect of EDU treatment on numbers of leaves, nodules and pods, and dry pod weight of mung bean plants (Mean ± 1SE) Plant age and treatment 30 DAS Non-treated EDU-treated 45 DAS Non-treated EDU-treated 60 DAS Non-treated EDU-treated 80 DAS Non-treated EDU-treated Parameters Leaf number 11.00 ± 0.58 13.00 ± 0.58 21.00 ± 0.58 26.00 ± 1.15 35.00 ± 1.53 42.67 ± 1.45 37.67 ± 0.88 44.00 ± 2.30 Number of nodules 0±0 0±0 6.00 ± 0.58 10.00 ± 1.15 29.00 ± 1.15 37.00 ± 2.08 39.67 ± 2.60 68.00 ± 2.89 Pod number 0±0 0±0 0±0 0±0 17.67 ± 1.20 26.00 ± 1.53 28.00 ± 2.08 42.67 ± 2.90 Dry pod weight (g) 0±0 0±0 0±0 0±0 1.59 ± 0.08 2.04 ± 0.07 5.35 ± 0.06 6.76 ± 0.43

content longer by regulating protein catabolism in leaves. Lee (1991) and Van Hove et al. (2001) observed a significant positive relationship between O3 sensitivity

of the tissue and ascorbic acid content. Increase in ascorbic acid content due to EDU-treatment was reported in other plants (Gillespie et al., 1998; Lyons et al., 2000).

Leaf area (cm2)

Dry root weight (g )

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(F)

0 1.5

Fresh root weight (g )

Root length (cm)

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0.72 0.68 0.40

RSR (g g-1)

0.1 0.30 0.0 400 0.20

(D)
0.2

300 200 100 0 30 45 60 80 30 45 60 80 0.0 0.1

DAS

DAS

Fig. 4. Effect of EDU treatment on RSR, LWR, SLA and NPP of mung bean plants.

day-1)

2.0 1.5 0.5

Non-treated EDU-treated

0.00225 0.00200 0.00100 0.00050 0.00000 900 750 600 400 300 200 100 0 21000 18000 15000 8000 4000

CGR (g cm-2 day-1)

0.0 0.005 0.004 0.003 0.002 0.001 0.24 0.22 0.20 0.04 0.02 0.00

(B)

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(F)

RGR (g day-1)

30-45

45-60

60-80

0

30-45

45-60

60-80

Time interval (DAS)

Time interval (DAS)

Fig. 5. Effect of EDU treatment on AGR, CGR, RGR, NAR, LAR and LAD of mung bean plants.

Perhaps, EDU application maintained a higher level of ascorbic acid by reducing production of free radicals in presence of O3. Increase in phenol content under O3 stress may be ascribed to its increased synthesis from amino acids due to

hydrolysis of protein or inhibition of protein synthesis (Ambasht and Agrawal, 2003). Booker and Miller (1998) found significant increase in average concentration of aqueous methanol soluble phenolics in leaves of soybean (Glycine max (L.) Merr.) at elevated O3. In

LAD (days)

LAR (cm2 g-1)

NAR (g cm-2 day-1)

2.5

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AGR (cm

NPP (g plant-1 day-1)

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LWR (g g-1)

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Seed weight pod-1 (g)

0.4 0.3

(A)

(B)

3

2 0.2 0.1 0.0 16 1

0

Harvest index (%)

12 20 8 4 0 10 0

Control

EDU-treated

Control

EDU-treated

Fig. 6. Effect of EDU treatment on yield parameters of mung bean plants.

Table 4 Variance ratio (one-way ANOVA) for yield parameters of moong Parameters Seed podÀ1 Yield plantÀ1 Yield mÀ2 Test weight Harvest index * = p < 0.05, significant. ** = p < 0.01, Significance level * * * ** NS *** = p < 0.001, NS = non-

the present investigation, lower phenol content in EDUtreated mung bean plants suggests that its application reduced the synthesis of phenolic compounds by alleviating the negative impact of O3 on proteins. The protective influence of EDU was further supported by enhancement in plant height, biomass accumulation, leaf area and yield of EDU-treated plants over non-EDU-treated ones. Protective effects of EDU against O3 have been widely reported from laboratory and field studies (Hassan et al., 1995; Brunschon-Harti et al., 1995a,b; Carnahan et al., 1978; Agrawal and Agrawal, 1999; Manning, 2000). Total stem height and basal diameter of Pinus taeda L. increased significantly due to fortnightly application of 450 ppm EDU as foliar spray (Manning et al., 2003). Brunschon-Harti et al. (1995a) found that increased O3 dose significantly decreased shoot biomass of P. vulgaris, whereas EDU-treated plants had higher biomass than control ones. Roberts et al. (1985), however, found a reduction in leaf dry weight under EDU treatment. Leaf biomass of Trifolium subterraneum plants increased under EDU treatment as compared to nonEDU-treated ones (Tonneijck and Van Dijk, 2002a).

Test weight (g)

(C)

(D)
30

Results of present study clearly indicated that ambient O3 concentrations at experimental site reduced plant height, total biomass and yield of non-EDU-treated plants as compared to EDU-treated ones. Ozone depressed plant biomass and crop yield in a variety of plant species (Krupa et al., 2001). Such reductions reflect a decline in carbon gain due to an inhibition of photosynthesis, which may be attributed to reduced carboxylation activity of Rubisco and/or a decrease in Rubisco quantity (Pell et al., 1997). Ozone exposure caused reduction in leaf number (Kasana, 1992; Tonneijck and Van Dijk, 1997) and leaf area (Bambridge et al., 1995). In the present study also a similar effect was observed in non-EDU-treated plants than EDUtreated ones. Increments in number of nodules in EDU-treated plants further suggest a protective role of EDU under elevated O3 levels. Various growth indices also modified due to EDUtreatment. RSR, LWR and NPP of EDU-treated plants increased over non-treated ones. A higher RSR in EDUtreated plants clearly suggests a protective role of EDU on biomass allocation to roots. Barnes et al. (1990) found significant negative correlation between RSR and O3 concentration. AGR represents the amount of growing material present. EDU-treatment led the plants to accumulate more biomass by alleviating the adverse effects of O3. Higher CGR and RGR in EDU-treated plants at early stages support the observations of Gillespie et al. (1998) in snap bean plants. Higher LAD (a measure of the persistence of assimilatory surface) of EDU-treated plants confirms the maintenance of more assimilatory surface than non-treated ones. Increases in number of pods and seeds podÀ1 and test weight due to EDU-treatment contributed to higher harvest index and seed yield of these plants as compared to non-EDU treated ones. The reduction in grain yield of winter wheat due to O3 has been ascribed to decrease in number of grains spikeletÀ1 or increase in number of infertile florets (Ollerenshaw and Lyons, 1999). EDU applied to the plants growing at higher O3 levels led to yield increments of 36% in navy bean (Hofstra et al., 1978), 24% in white bean (Temple and Bisessar, 1979), 30% in tomato (Legassicke and Ormrod, 1981), 35% in potato (Bisessar, 1982) and 20% in soybean (Damicone, 1985). Kostca-Rick and Manning (1993), however, found a loss in bush bean pod weight upon EDU treatment, suggesting that the response of EDU depends upon applied O3 concentration. EDU treatment increased the pod weight by 49% in charcoal filtered and 65% in charcoal filtered plus additional O3 treatments (Brunschon-Harti et al., 1995a). Smith et al. (1987), however, did not find significant yield differences between EDU-treated and non-treated soybean cultivars. Tonneijck and Van Dijk (2002a) found that yield of green and mature pods reduced in non-EDU-treated P. vulgaris plants.

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The mechanism of action of EDU in alleviating O3 effects is suggested to be biochemical and not biophysical (Bennett et al., 1978; Lee and Bennett, 1982; Hassan et al., 1995). EDU detoxifies O3 in apoplastic region of the cells and does not act directly as an antioxidant. EDU helps in maintaining higher levels of cellular antioxidants associated with protection during O3 stress (Lyons et al., 2000). The results on EDU induced maintenance of higher levels of antioxidants and activities of protective enzymes in plants during O3 stress are inconsistent (Brunschon-Harti et al., 1995b; Lee et al., 1997). The present study clearly showed that O3 concentrations present in suburban area of Allahabad city cause negative effect on growth, biomass accumulation and allocation and yield of mung bean plants. Application of EDU as soil drench has protected the plants against O3 damage. EDU induced tolerance to O3 may be ascribed to its ability in maintaining of higher levels of photosynthetic pigments, protein and ascorbic acid contents in plants. The study confirms the usefulness of EDU as a tool for assessing the phytotoxic concentrations of O3 under ambient field conditions. Such studies are especially important for tropical country, like India where meteorological conditions are favourable for the formation of O3.

Acknowledgement Authors wish to express sincere thanks to C. S. I. R. (New Delhi) for financial support, to Prof. R.B. Lal, Vice chancellor, Allahabad Agricultural Institute (DU) and Prof. P.W. Ramteke, Director (Research) for providing laboratory facilities and encouragements. Authors are also grateful to Prof. W.J. Manning, University of Massachusetts, Amherst, USA for providing EDU as gift and to Profs. Madhoolika Agrawal, B.R. Chaudhary (B.H.U.) and both anonymous reviewers for their comments and fruitful suggestions.

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